Version Compatibility

Reference examples tested with: DADA2 1.30+, QIIME2 2024.2+, phyloseq 1.46+, scanpy 1.10+, scikit-learn 1.4+

Before using code patterns, verify installed versions match. If versions differ:

• R: packageVersion('<pkg>') then ?function_name to verify parameters
• CLI: <tool> --version then <tool> --help to confirm flags

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

Taxonomy Assignment

"Assign taxonomy to my ASVs" → Classify amplicon sequence variants against reference databases (SILVA, GTDB, UNITE) using naive Bayes or exact-matching approaches for taxonomic annotation.

• R: dada2::assignTaxonomy() with SILVA/GTDB reference
• CLI: qiime feature-classifier classify-sklearn for QIIME2 workflows

DADA2 Naive Bayes Classifier

library(dada2)

seqtab_nochim <- readRDS('seqtab_nochim.rds')

# SILVA for 16S (download from https://zenodo.org/record/4587955)
taxa <- assignTaxonomy(seqtab_nochim, 'silva_nr99_v138.1_train_set.fa.gz',
                       multithread = TRUE)

# Add species-level (exact matching)
taxa <- addSpecies(taxa, 'silva_species_assignment_v138.1.fa.gz')

# Check results
head(taxa)

GTDB for 16S

# GTDB-formatted database (better for environmental samples)
taxa_gtdb <- assignTaxonomy(seqtab_nochim, 'GTDB_bac120_arc53_ssu_r220_fullTaxo.fa.gz',
                            multithread = TRUE)

UNITE for ITS (Fungi)

# UNITE database for fungal ITS
taxa_its <- assignTaxonomy(seqtab_nochim, 'sh_general_release_dynamic_25.07.2023.fasta',
                           multithread = TRUE)

QIIME2 Feature Classifier

# Train classifier (one-time)
qiime feature-classifier fit-classifier-naive-bayes \
    --i-reference-reads silva-138-99-seqs.qza \
    --i-reference-taxonomy silva-138-99-tax.qza \
    --o-classifier silva-138-99-nb-classifier.qza

# Classify ASVs
qiime feature-classifier classify-sklearn \
    --i-classifier silva-138-99-nb-classifier.qza \
    --i-reads rep-seqs.qza \
    --o-classification taxonomy.qza

VSEARCH Exact Matching

# Faster but requires exact or near-exact matches
vsearch --usearch_global asv_seqs.fasta \
    --db silva_138_SSURef_NR99.fasta \
    --id 0.97 \
    --blast6out taxonomy_vsearch.tsv \
    --top_hits_only

RDP Classifier

library(dada2)

# RDP training set (less detailed than SILVA)
taxa_rdp <- assignTaxonomy(seqtab_nochim, 'rdp_train_set_18.fa.gz',
                           multithread = TRUE)

IDTAXA (DECIPHER) - Often More Accurate

Goal: Classify ASVs using DECIPHER's tree-based IDTAXA classifier, which provides more conservative and often more accurate assignments than naive Bayes.

Approach: Convert ASV sequences to DNAStringSet, classify against a pre-trained IDTAXA model, and convert the hierarchical output to a standard taxonomy matrix.

library(DECIPHER)

# Load IDTAXA training set (download from http://www2.decipher.codes/Downloads.html)
load('SILVA_SSU_r138_2019.RData')  # Creates 'trainingSet' object

# Convert ASV sequences to DNAStringSet
dna <- DNAStringSet(getSequences(seqtab_nochim))

# Classify with IDTAXA
ids <- IdTaxa(dna, trainingSet, strand = 'top', processors = NULL, verbose = TRUE)

# Convert to matrix format like assignTaxonomy
ranks <- c('domain', 'phylum', 'class', 'order', 'family', 'genus', 'species')
taxa_idtaxa <- t(sapply(ids, function(x) {
    m <- match(ranks, x$rank)
    taxa <- x$taxon[m]
    taxa[startsWith(taxa, 'unclassified_')] <- NA
    taxa
}))
colnames(taxa_idtaxa) <- ranks

Confidence Filtering

# assignTaxonomy returns bootstrap confidence
# Filter low-confidence assignments
taxa_filtered <- taxa
taxa_filtered[taxa_filtered < 80] <- NA  # If using minBoot output

# Or use confidence threshold during assignment
taxa <- assignTaxonomy(seqtab_nochim, 'silva_nr99_v138.1_train_set.fa.gz',
                       minBoot = 80, multithread = TRUE)

Combine into phyloseq

library(phyloseq)

# Create phyloseq object
ps <- phyloseq(otu_table(seqtab_nochim, taxa_are_rows = FALSE),
               tax_table(taxa))

# Add sample metadata
sample_data(ps) <- read.csv('sample_metadata.csv', row.names = 1)

# Rename ASVs for readability
taxa_names(ps) <- paste0('ASV', seq(ntaxa(ps)))

Database Comparison

Database	Organisms	Taxonomy	Updated
SILVA 138.1	Bacteria, Archaea, Eukaryotes	7 ranks	2024
GTDB R220	Bacteria, Archaea	7 ranks (genome-based)	2024
RDP 18	Bacteria, Archaea	6 ranks	2016
UNITE 10.0	Fungi	7 ranks	2024
PR2 5.0	Protists	8 ranks	2024

Related Skills

• amplicon-processing - Generate ASV table for classification
• diversity-analysis - Analyze classified communities
• metagenomics/kraken-classification - Read-level taxonomic classification